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| United States Patent |
5,162,368
|
|
Franco
, et al.
|
November 10, 1992
|
Butalactin and its use as pharmaceutical
Abstract
Butalactin, a compound of the formula I
##STR1##
can be produced by cultivation of Streptomyces species Y-86,36923.
Butalactin has an antibiotic activity.
| Inventors:
|
Franco; Christopher M. M. (Bombay, IN);
Vijayakumar; Erra K. (Bombay, IN);
Chatterjee; Sugata (Bombay, IN);
Ganguli; Bimal N. (Bombay, IN);
Blumbach; Jurgen (Bombay, IN)
|
| Assignee:
|
Hoechst Aktiengesellschaft (Frankfurt am Main, DE)
|
| Appl. No.:
|
563188 |
| Filed:
|
August 6, 1990 |
Foreign Application Priority Data
| Current U.S. Class: |
514/473; 549/318 |
| Intern'l Class: |
A61K 031/34; C07D 307/33 |
| Field of Search: |
549/318
514/473
|
References Cited
U.S. Patent Documents
| 4070480 | Jan., 1978 | Aldridge et al. | 514/473.
|
| 4145437 | Mar., 1979 | Aldridge et al. | 514/473.
|
| 4188331 | Feb., 1980 | Pernet et al. | 549/318.
|
Other References
"A New Inducer of Anthracycline Biosynthesis" from Streptomyces
Viridochromogenes, The Journal of Antibiotics, vol. 35, No. 12, pp.
1722-1723.
|
Primary Examiner: Ivy; C. Warren
Assistant Examiner: Owens; Amelia A.
Attorney, Agent or Firm: Finnegan, Henderson, Farabow, Garrett and Dunner
Claims
We claim:
1. Butalactin, a compound of the formula I
##STR3##
2. An antibiotic pharmaceutical composition comprising an antibiotically
effective amount of the compound according to claim 1 together with a
pharmaceutically acceptable carrier.
3. A method of controlling bacterial activity comprising administering to a
host in need of such treatment an effective amount of the compound
according to claim 1 together with a pharmaceutically acceptable carrier.
Description
DESCRIPTION
This invention relates to a compound named Butalactin, a process for its
production by cultivating species culture number HIL Y-86,36923 (Str. sp.
Y-86,36923) or its mutants or variants as well as the use of Butalactin as
a pharmaceutical and a biosynthetic regulator.
Str. sp. Y-86,36923 was isolated from soil collected at Pune, Maharashtra,
India and it has been deposited with the Deutsche Sammlung von
Mikroorganismen under the conditions of the treaty of Budapest on May 25,
1989 (DSM 5372). Variants and mutants of Str. sp. Y-86,36923 can be
obtained in a known manner using a mutagen, such as
N-methyl-N-nitro-N'-nitrosoguanidine or ultraviolet light. The
microorganism Str. sp. Y-86,36923 belongs to the order Actinomycetales,
family Streptomycetaceae and genus Streptomyces.
Str. sp. Y-86,36923 is considered to be a new strain since it differs from
the known strains in some of its morphological, cultural and physiological
characteristics as will be clear from the description hereinafter. It is
considered to be a new strain also because it produces a new antibiotic
compound herein called Butalactin as will be clear from the description
hereinafter.
As is apparent in the following detailed description of the invention,
Butalactin of this invention is a 2,3-disubstituted butanolide antibiotic,
but differs from all known disubstituted butanolide metabolites such as
A-factor (Bioorg. Khim. 2, 1142-1147, 1976), anthracycline-inducing
factors (J. Antibiotics, 35, 1722-1723, 1982; Biotechnology Lett. 5,
591-596, 1983), Virginiae butanolides (J. Antibiotics, 40, 496-504, 1987)
and various racemic analogues (J. Antibiotics, 41, 1828-1837, 1988).
Butalactin is a compound of the formula I
##STR2##
According to IUPAC nomenclature the chemical name of the compound of this
invention is
2-(4',5'-epoxy-1'-oxo-2'(E)-hexen)-yl-2-hydroxy-3-hydroxymethyl-2,3-(Z)-bu
tanolide or, alternatively,
2-(4',5'-epoxy-hex-2'(E)-en)oyl-2-hydroxy-4-hydroxymethyl-2,3-(Z)-butanoli
de.
Chemical Abstract Service (CAS)-Online-Literature Search performed with the
search keys of molecular weight and molecular formula also confirmed the
fact that Butalactin is a new compound.
The novel antibiotic of this invention is active in vitro against a number
of Gram-positive and Gram-negative microorganisms. It is also active in
the regulation of biosythesis of secondary metabolites. Accordingly, it
may be used as a therapeutic drug and as a biochemical regulator in the
control of antibiotic biosynthesis.
According to the present invention there is also provided a process for the
isolation of Str. sp. Y-86,36923 from soil using a nutrient medium at a pH
of 6.5 to 8.5 in a known manner.
The nutrient medium used for isolation of the microorganism from soil
consists of carbon and nitrogen sources, inorganic nutrient salts and
solidifying agents. Sources of carbon may, for example, be glucose,
starch, dextrin, glycerol, sucrose or molasses. Sources of nitrogen may,
for example, be peptone, yeast extract, beef extract, malt extract, casein
or amino acids such as arginine or asparagine. The solidifying agent may,
for example, be agar. The inorganic nutrient salts may, for example, be
salts of sodium, potassium, magnesium, calcium, phosphorus or sulfur.
The microorganism of this invention elongates colourless aerial mycelia
from branched substrate mycelia. Spore chains are formed in spirals on top
of aerial mycelia. The spirals are open. Short spore chains representative
of Section RA are also common. Neither whirl or ascospores are observed.
Mature spore chains contain 10-20 spores per chain. The cultural
characteristics of the microorganism on various agar media are described
hereinbelow:
1. Yeast extract: malt extract agar
Growth: good, wrinkled, dry
Aerial mycelium: scant, powdery, light grey
Reverse: yellowish white
Soluble pigment: none
2. Oatmeal agar
Growth: good, flat, dry
Aerial mycelium: moderate, powdery, white
Reverse: pale yellow
Soluble pigment: none
3. Inorganic salts: starch agar
Growth: good, raised, dry
Aerial mycelium: good, powdery, grey
Reverse: grey-olive
Soluble pigment: none
4. Glycerol: asparagine agar
Growth: good, wrinkled, dry
Aerial mycelium: weak, powdery, white
Reverse: pale yellow
Soluble pigment: none
5. Peptone: yeast extract--iron agar
Growth: moderate, flat, sand-like
Aerial mycelium: none
Reverse: pale brown
Soluble pigment: none
6. Tyrosine agar
Growth: good, wrinkled, dry
Aerial mycelium: none
Reverse: light yellow
Soluble pigment: none
7. Sucrose: nitrate agar
Growth: good, wrinkled, sand-like
Aerial mycelium: scant, powdery, whitish
Reverse: pale yellow
Soluble pigment: none
8. Glucose: asparagine agar
Growth: good, wrinkled, dry
Aerial mycelium: weak, powdery, light grey
Reverse: pale yellow
Soluble pigment: none
The optimum growth temperature range for the microorganism of this
invention is from 25.degree. C. to 37.degree. C. This microorganism
liquefies gelatin in glucose-peptone-gelatine medium, hydrolyzes starch in
starch-inorganic salts agar, coagulates skimmed milk, does not form
H.sub.2 S, and does not reduce nitrate. Str. sp. Y-86,36923 grows well on
Czapek's solution agar.
No production of melanoid pigment is observed in tyrosine agar,
peptone-yeast extract--iron agar or tryptone--yeast extract broth.
The carbon source assimilation pattern of this microorganism is as follows
(in Pridham-Gottlieb's medium):
Positive: D-glucose, L-arabinose, D-xylose, m-inositol, D-mannitol,
D-fructose, galactose, maltose, cellobiose, Na-glutamate, mannose,
lactose, sucrose.
Weak: Rhamnose, raffinose.
Negative: Cellulose.
Str. sp. Y-86,36923 is inhibited by streptomycin at concentrations greater
than 6.25 .mu.g/ml, can tolerate NaCl at concentrations greater than 6%
but less than 7%, and has a pH tolerance range of 6.0-9.0.
Str. sp. Y-86,36923 differs substantially from the known microorganisms
which produce Cineromycin B and the related compound Albocycline. These
are Streptomyces cinerochromoges, Streptomyces brunneogriseus,
Streptomyces roseocinereus, and Streptomyces roseochromogenes var
albocyclini.
The published information on the cultural and physiological characteristics
of other known microorganisms show clear difference when compared with the
microorganism of this invention.
In addition, when Str. sp. Y-86,36923 is fermented it produces the novel
antibiotic Butalactin, in addition to the known antibiotic Cineromycin B.
From the above observations it is evident that the microorganism of this
invention is a new species of Streptomyces.
It may be well understood to those skilled in the art that this invention
is not limited to the particular organism which has been specified above
but includes all those spontaneous and artificial mutants and variants
derived from the said microorganism which are capable of producing the new
antibiotic Butalactin.
A further subject of the present invention is a process for the production
of Butalactin, said process comprising cultivating Str. sp. Y-86,36923 by
fermentation at a pH between 6.0 and 9.0, preferably between 6 and 7, and
a temperature between 18.degree. and 40.degree. C., preferably between
20.degree. and 37.degree. C., under aerobic conditions in a nutrient
medium containing sources of carbon and nitrogen, nutrient inorganic
salts, and trace elements, and isolating the compound from the culture
broth in a known manner such as herein described.
The carbon sources used in the nutrient medium for production of the novel
antibiotics may, for example, be glucose, starch, dextrin, glycerol,
sucrose, molasses or oil. Sources of nitrogen used in the nutrient medium
for production of the novel antibiotics may, for example, be soyabean
meal, yeast extract, beef extract, malt extract, cornsteep liquor,
peptone, gelatin or casein. Nutrient inorganic salts/mineral salts used in
the nutrient medium for production of the novel antibiotics may, for
example, be sodium chloride, magnesium sulfate, ammonium sulfate or
calcium carbonate. As trace elements, for example, iron, manganese,
copper, zinc or cobalt may be used.
Preferably Str. sp. Y-86,36923 is cultivated at about 27.degree. C.
(.+-.1.degree. C.) and pH about 7.0. The fermentation is preferably
stopped after 23 to 48 hours when maximum yields of the compounds are
obtained. The fermentation may, preferably, be a submerged fermentation.
The process of fermentation and formation of Butalactin can be monitored
by the antibacterial activity of the culture fluid and mycelium against
Staphylococcus aureus 209 P and against Escherichia coli 9632 in agar
medium, and by thin layer chromatography on silica gel plates with ethyl
acetate as developing solvent.
If desired, an antifoaming agent such as Desmophen.RTM. (Polyols, Bayer AG,
Leverkusen, Germany) may be used in the nutrient medium during
fermentation of the culture.
Butalactin can be obtained from the culture broth by extraction, preferably
with a water-immiscible solvent after the pH has been adjusted to 6.5 to
7.5. The solvents could be ethyl acetate or chloroform; preferably it is
ethyl acetate and the preferred pH is about 7.0. The solvent extract is
concentrated to remove the solvent and then chromatographed further.
Butalactin can also be obtained from the culture broth by direct
adsorption on suitable absorbents such as Amberlite.RTM. XAD-4 or 7
(Porous adsorbent resin based on polystyrene or acrylic acid ester, Rohm
and Haas Co., U.S.A.), or Diaion.RTM. HP-20 (high porosity adsorbent resin
based on a polystyrenedivinylbenzene copolymer, Mitsubishi Chemical
Industries, Japan); the preferred adsorbent being Diaion.RTM. HP-20. The
compound according to the invention is eluted from the adsorbent using
appropriate mobile phases, such as chloroform, methanol or acetone, either
singly, in combination with each other, or with water, and the eluates are
then evaporated to dryness. The preferred eluant is methanol. The active
eluates thus obtained are pooled and concentrated.
The aforementioned concentrated eluates or extracts containing Butalactin,
can be further purified in a number of ways. For example, re-adsorption
and elution processes with activated carbon, Amberlite.RTM. XAD-4 and 7,
Diaion.RTM. HP-20, gel filtration with Sephadex.RTM. LH-20 gel (gel of
defined porosity on agarose, Pharmacia Fine Chemicals AB, Sweden) and its
equivalents; adsorption chromatography on alumina and silica gel, can be
conveniently combined for further purification. In addition, thin-layer
chromatography, medium-pressure and high-pressure liquid chromatography
using suitable adsorbents such as silica gel and modified silica
gel-C.sub.18 with suitable solvent systems may be used. Furthermore,
counter current chromatography with a particular solvent system may work
well for the said purpose. Preferably, repeated silica gel chromatography
with ethyl acetate and petroleum ether (40.degree. to 60.degree. C.) as
the eluting solvents is used. During this chromatography the known
compound Cineromycin B is also separated out.
Butalactin is a pale yellow, thick syrup at room temperature (25.degree.
C.). It is soluble in water, methanol, ethanol, propylene glycol,
dimethylsulfoxide, methylene chloride, ethyl acetate and chloroform. It is
insoluble in hexane and petroleum ether (40.degree. to 60.degree. C.).
In the thin layer chromatography (TLC) systems indicated below Butalactin
has the following values:
TLC plate: Precoated silica gel plate: Article No. 5544 from E. Merck,
Darmstadt.
______________________________________
Rf of EtOAc
______________________________________
Butalactin 0.44
Cineromycin B 0.51
______________________________________
BRIEF DESCRIPTION OF DRAWINGS
The analytical high-pressure liquid chromatography (HPLC) is shown in FIG.
1. HPLC was carried out using the following:
Column packing: ODS-Hypersil.RTM.-10 .mu., 4.times.(30+100) mm (Column
material based on octadecyl silan, Shandon Labortechnik, Frankfurt/Main,
Germany)
Flow Rate: 0.5 ml/min
Detection: 240 nm
Solvent: MeOH: Water (1:3)
The spectroscopic data of Butalactin is listed below:
1. UV max in methanol and in 0.1 N HCl-methanol is 242 nm, in 0.1 N
NaOH-methanol it shifts to 206 nm. The absorption spectrum was measured in
the range of 200 to 800 nm using a Uvikon 810 Spectrophotometer.
2. The IR spectrum (neat) was determined using a Perkin Elmer P.E. 521 IR
Spectrometer--see FIG. 2.
3. The .sup.1 H-NMR spectrum was determined on a 90 MHz JEOL FX-90Q
instrument using CDCl.sub.3 as solvent--see FIG. 3.
4. The .sup.13 C-NMR spectrum was determined on a 90 MHz JEOL FX-90Q
instrument using CDCl.sub.3 as solvent--see FIG. 4 of the accompanying
drawings.
5. Mass Spectroscopy was carried out on a VG ZAB 3F instrument using
Electron Impact (EI), Chemical Ionization (CI), and Fast Atom Bombardment
(FAB) modes of ionization. The Molecular weight was found to be 242.0857
which corresponds to a Molecular formula of C.sub.11 H.sub.14 O.sub.6.
The above data together with the chemical analysis indicates that
Butalactin has a structure as shown in formula I.
Butalactin is active against gram-positive and gram-negative bacteria. When
tested by the agar well method in Antibiotic Assay medium it has an
activity which is shown in Table I below:
TABLE I
______________________________________
Concentration
Test Organism 2 mg/ml 1 mg/ml
______________________________________
* *
1. Staphylococcus aureus 209 P
21 19
2. Staphylococcus epidermidis 823
16 14
3. Staphylococcus haemolyticus 809
16 13
4. Streptococcus faecalis 21777
17 15
5. Streptococcus faecalis Eder
23 21
6. Escherichia coli 9632
21 16
7. Enterobacter cloacae
14 13
8. Citrobacter freundii
17 15
9. Citrobacter 2046 E 16 14
10. Proteus vulgaris 15/21 12/20
11. Proteus mirabilis 18 16
12. Pseudomonas aeruginosa
-- --
13. Candida albicans -- --
14. Aspergillus niger -- --
______________________________________
* zone of inhibition in mm
In addition to the antibacterial activity Butalactin was also found to
inhibit the biosynthesis of secondary metabolites, such as antibiotics, at
concentrations that do not inhibit growth of the producing organism.
The invention will be further illustrated by preferred examples, but should
not be considered as limited by those examples.
EXAMPLE I
Isolation of Streptomyces sp. Y-86,36923 from soil
(a) Preparation of nutrient isolation media
______________________________________
Medium 1: Glucose 1.0 g
Glycerol 1.0 g
L-arginine 0.3 g
K.sub.2 HPO.sub.4
0.3 g
MgSO.sub.4 .times. 7H.sub.2 O
0.2 g
NaCl 0.3 g
Yeast extract 2.0 g
FeSO.sub.4 .times. 7H.sub.2 O
10.0 mg
CuSO.sub.4 .times. 5H.sub.2 O
1.0 mg
ZnSO.sub.4 .times. 7H.sub.2 O
1.0 mg
MnSO.sub.4 .times. 7H.sub.2 O
1.0 mg
Agar 15.0 g
Distilled water
1 liter
pH 6.5
Medium 2: Glucose 2.0 g
L-asparagine 1.0 g
K.sub.2 HPO.sub.4
0.5 g
MgSO.sub.4 .times. 7H.sub.2 O
0.5 g
Soil Extract 200 ml
Agar 15.0 g
Distilled water
800 ml
pH 8.0
Medium 3: Starch 10.0 g
Casein 0.3 g
KNO.sub.3 2.0 g
NaCl 2.0 g
K.sub.2 HPO.sub.4
2.0 g
MgSO.sub.3 .times. 7H.sub.2 O
0.05 g
CaCO.sub.3 0.02 g
FeSO.sub.4 .times. 7H.sub.2 O
0.01 g
Agar 15.0 g
Distilled water
1 liter
pH 7.2-7.5
______________________________________
The media were sterilised at 121.degree. C. for 30 minutes. In allcases,
the sterilized media were cooled to 45.degree. C., poured into petri
plates and allowed to solidify.
(b) Preparation of soil suspension
One gram of soil was heated in a hot air oven to 110.degree. .C for one
hour. After cooling it was suspended in distilled water and shaken well.
The soil was allowed to settle and the supernatent fluid was used to
inoculate each one of the above mentioned media at a time.
(c) Inoculation of the isolation medium
One ml of the soil suspension was inoculated onto petri dishes containing
50 ml of any of the above mentioned nutrient isolation media.
(d) Isolation of Streptomyces sp. Y-86,36923
The inoculated petri dish was incubated at 37.degree. C. for 10 days and
Streptomyces sp. Y-86,36923 isolated from among the growing
microorganisms.
EXAMPLE II
Cultivation of Streptomyces sp. Y-86,36923 for the fermantative production
of Butalactin
Streptomyces sp. Y-86,36923 was maintained on yeast extract-malt extract
having the following composition:
______________________________________
Malt extract 10.0 g
Yeast extract 4.0 g
Glucose 4.0 g
Agar 15.0 g
Distilled Water 1 liter
pH 7.0
______________________________________
The medium was distributed in test tubes and sterilized at 121.degree. C.
for 30 minutes. The tubes were cooled in a slanting position for
preparation of agar slants. The slants were inoculated with the culture
and incubated at 28.degree. C. for 10 to 15 days when good growth and
sporulation were observed. A suspension of the spores in distilled water
from one slant was used to inoculate five 500 ml Erlenmeyer flasks each
containing 100 ml of the seed culture medium.
Composition of the seed culture medium
______________________________________
Glucose 15.0 g
Soyabean meal 15.0 g
Cornsteep liquor 5.0 g
CaCO.sub.3 2.0 g
NaCl 5.0 g
Distilled water 1 liter
pH 6.5
______________________________________
The above medium was distributed in 100 ml amounts in 500 ml Erlenmeyer
flasks and sterilized at 121.degree. C. for 30 minutes. The flasks were
cooled, inoculated with spore suspension or mycelial plugs and shaken at
240 r.p.m. for 72 hours at 27.degree. C. (.+-.1.degree. C.) on a rotary
shaker with 1.5 inch throw. The resultant growth was used to inoculate two
hundred 500 ml flasks each containing 100 ml of the production culture
medium at 2 to 4% (v/v).
Composition of the production medium
______________________________________
Glucose 10.0 g
Soluble starch 10.0 g
Malt extract 7.5 g
Peptone 7.5 g
MgSO.sub.4 .times. 7H.sub.2 O
1.0 g
NaCl 3.0 g
CuSO.sub.4 .times. 5H.sub.2 O
7.0 mg
FeSO.sub.4 .times. 7H.sub.2 O
1.0 mg
MnCl.sub.2 .times. 4H.sub.2 O
8.0 mg
ZnSO.sub.4 .times. 7H.sub.2 O
2.0 mg
Distilled water 1 liter
pH 6.5
______________________________________
The fermentation was carried out at 27.degree. C. (.+-.1.degree. C.) on a
rotary shaker at 240 r.p.m. with a 1.5 inch throw. When fermentation was
discontinued at the end of 45 to 48 hours, the diameter of the zone of
inhibition versus Staphyloccus aureus 209 P was 16 mm and versus
Escherichia coli as 13 mm, when the culture filtrate was tested by the
agar well (6 mm diameter) method and the pH of the culture fluid ranged
from 6.8 to 7.0. The packed cell volume was 20% (v/v). The harvested
culture broth containing the antibiotic was centrifuged to separate the
mycelium and the culture fluid and further processed as described in
Example IV.
EXAMPLE III
Cultivation of Streptomyces sp. Y-86,36923 for the fermentative production
of Butalactin
The procedure of Example II was repeated with the following differences:
Str sp. Y-86,36923 was grown on an agar medium with the following
composition:
______________________________________
Soluble starch 10.0 g
K.sub.2 HPO.sub.4 1.0 g
MgSO.sub.4 .times. 7H.sub.2 O
1.0 g
NaCl 1.0 g
(NH.sub.4).sub.2 SO.sub.4
2.0 g
CaCO.sub.3 2.0 g
FeSO.sub.4 .times. 7H.sub.2 O
0.1 mg
MnCl.sub.2 .times. 4H.sub.2 O
0.1 mg
ZnSO.sub.4 .times. 7H.sub.2 O
0.1 mg
Agar 15.0 g
Distilled water 1 liter
pH 7.2
______________________________________
The composition of the seed culture medium is similar to that in Example
II.
Composition of production medium
______________________________________
Glucose 20.0 g
Peptone 5.0 g
Beef extract 5.0 g
CaCO.sub.3 3.0 g
Distilled water 1 liter
pH 7.0
______________________________________
2 ml of Desmophen.RTM. was added as antifoam agent. 10 l of the above
medium were taken in a 15 l fermenter. The medium was sterilized trough
direct and indirect steam for 20 minutes at 121.degree. C. The fermenter
was cooled and inoculated with seed culture (9% v/v). The fermentation was
carried out at 27.degree. C. (.+-.1.degree. C.) under stirred conditions
at 120 r.p.m. with aeration at a rate of 60 to 70 liters per minute. When
fermentation was discontinued at the end of 23 to 27 hours the pH of the
culture broth was pH 7.4 and the diameter of the zone of inhibition versus
Staphylococcus aureus 209 P was 22 mm and versus Escherichia coli was 14
mm when the culture filtrate was tested by the agar well method (6 mm
diameter). The packed cell colume was 15% (v/v). The culture broth was
processed as in Example V.
EXAMPLE IV
Isolation and purification of Butalactin
Approximately 16 liters of the culture filtrate, as obtained from Example
II, was extracted twice with 10 liters each of ethyl acetate after
adjusting the pH to 7.0. The aqueous layers were discarded and the
combined ethyl acetate extracts where evaporated under vacuum to dryness.
Approximately 6.4 g of crude extract was obtained. This crude extract was
dry-charged onto a 5.times.20 cm silica gel (230-400 mesh size) column and
eluted starting with a Petroleum ether (40.degree.-60.degree. C.) ethyl
acetate mixture (1:1) followed by a stepwise (10%) increase in the ethyl
acetate concentration to 100%. This procedure yielded 1.7 g of Fraction A,
which eluted out at when the petroleum ether-ethylacetate gradient was
1:1, and 3.2 g of Fraction B which eluted out when the petroleum
ether-ethylacetate gradient was 3:7. Fraction A was further purified by
charging it into a 5.times.25 cm silica gel (230-400 mesh size) column
followed by elution with a CHCl.sub.3 -EtOAc (1:1) to (3:7) gradient. The
active fractions which were obtained with a CHCl.sub.3 -EtOAc (8:2)
concentration were concentrated and chromatographed on a 2.4.times.90 cm
Sephadex.RTM. LH 20 column in methanol to yield 400 mg of antibiotic
compound. This was further chromatographed on a 3.times.35 cm MPLC silica
gel (30 .mu.) column and eluted at a flow rate of 10 ml/min with a
chloroform to 2% methanol in chloroform stepwise (0.5%) gradient. When the
methanol concentration was between 1-1.5% the active compound was eluted
out and concentrated to yield 150 mg of a pure compound which was
identified as the known antibiotic Cineromycin B.
Fraction B was charged, in 3 equal lots, onto 4.5.times.45 cm MPLC silica
gel (30 .mu.) columns which were eluted with a petroleum
ether-ethylacetate (5:5) to (3:7) gradient. The flow rate was maintained
at 15 ml/min and the elution was monitored through a Knauer UV detector at
240 nm. Butalactin elutes out with a (4:6) petroleum ether-ethylacetate
mixture. The active fractions were concentrated under vacuum to give 650
mg pure compound.
EXAMPLE V
Isolation and purification of Butalactin
Culture filtrates from two fermentor batches, as outlined in Example III,
were pooled to give a volume of 17 liters. This was passed through a
column containing 1 liter of Diaion.RTM. HP-20; the column was washed with
10 liters of demineralized water and eluted with 5 liters of methanol. The
active methanol eluates were concentrated in vacuo to dryness to give 8.8
g crude product. This was charged onto two 5.times.25 cm silica gel
(230-400 mesh) columns, eluted with a chloroform-ethyl acetate (9:1) to
(3:7) gradient to give two active fractions--Fraction A (2.3 g),
containing Cineromycin B, which eluted out with chloroformethylacetate
(8:2); and Fraction B (4.3 g), containing Butalactin, which eluted out
when the chloroformethylacetate ratio was 5:5. Fraction B was purified in
two lots by repeated chromatography on 5.times.60 cm silica gel (30 .mu.)
MPLC colums with a petroleum ether-ethyl acetate (5:5) to (3:7) gradient.
The flow rate was maintained at 20 ml/min and monitoring of elution was
done with a Knauer UV detector at 240 nm. Concentration of the active
eluates, which eluted out when the petroleum ether-ethyl acetate ratio was
4:6, yielded 550 mg pure compound.
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